WO2025137751A1 - Polynucleotide, chimeric antigen receptor, vector, composition, method for producing a modified immune effector cell and use of the polynucleotide - Google Patents

Abstract

The present invention relates to a polynucleotide encoding an anti-BCMA chimeric antigen receptor (CAR) and to the polypeptide corresponding to the anti-BCMA chimeric antigen receptor (CAR) itself. The present invention also relates to a vector and a composition, comprising an immune effector cell, comprising the polynucleotide, as well as a method for producing the modified immune effector cell and the use of the polynucleotide, the vector, the composition or the immune effector cell produced by the method for the manufacture of a drug for the treatment of multiple myeloma.

Description

POLYNUCLEOTIDE, CHIMERIC ANTIGEN RECEPTOR, VECTOR, COMPOSITION, METHOD FOR PRODUCTION OF MODIFIED EFFECTOR IMMUNE CELL AND USE OF POLYNUCLEOTIDE

FIELD OF INVENTION

The present invention relates to a polynucleotide encoding an anti-BCMA chimeric antigen receptor (CAR) and the polypeptide corresponding to the anti-BCMA chimeric antigen receptor (CAR) itself. The present invention also relates to a vector and a composition comprising an effector immune cell comprising the polynucleotide, in addition to a method for producing a modified effector immune cell and the use of the polynucleotide, the vector, the composition or the effector immune cell produced by the method for the manufacture of a medicament for the treatment of multiple myeloma.

BACKGROUND OF THE INVENTION

Multiple myeloma (MM) is a type of hematologic cancer that affects plasma cells in the bone marrow (BM) and the disease is characterized by the proliferation of malignant plasma cells, which produce defective and/or excess monoclonal immunoglobulins, even in the absence of stimulating antigens, resulting in dysfunctions in multiple organs (Palumbo A., 2011; Feng, D., 2020).

Most patients with MM initially present with monoclonal gammopathy of undetermined significance (MGUS), characterized by the presence of monoclonal protein (M protein) in the blood. One to two percent of MGUS cases progress to MM per year (Atkin et al., 2018; Kyle et al., 2018). The intermediate stage between MGUS and MM is characterized as smoldering multiple myeloma (SMM). At this stage, patients have greater changes in blood M protein and clonal plasma cell counts but do not yet have symptoms. Approximately 10% of patients progress to MM per year (Ravindran et al., 2016). The diagnosis of multiple myeloma is defined when one or more symptoms are observed, including hypercalcemia, renal failure, anemia, elevated serum M protein and lytic bone lesions, and 10% or more plasma cells in the bone marrow or plasmacytoma identified by biopsy (Rajkumar et al., 2014; Rajkumar, SV, 2022).

According to statistics from the Global Cancer Observatory (GCO), in 2018 there were approximately 160,000 cases of multiple myeloma worldwide, representing approximately 1% of neoplastic diseases and 13% of hematologic cancers. The young population is rarely affected, with the average age at diagnosis being 70 years. In Brazil, the incidence of approximately 3,500 cases was recorded in 2012, and it is estimated that approximately 30,000 patients with multiple myeloma are undergoing treatment (da Silva Ferreira et al., 2017; Padala et al., 2021).

However, multiple myeloma is currently considered an incurable disease. Therefore, the primary goal of treatment is to increase survival and quality of life of patients. Treatment options aim to reduce the abundance of malignant plasma cells in BM and include procedures such as hematopoietic stem cell transplantation (HSCT), radiotherapy, and chemotherapy. In the last decade, the emergence of new treatment options, such as ^2nd generation proteasome inhibitors, ^2nd generation immunomodulatory drugs, and anti-CD38 monoclonal antibodies, have resulted in a significant improvement in the survival of patients with multiple myeloma (Cowan et al., 2022; Rodríguez-Otero et al., 2020).

However, the disease has a relapsing and progressive profile, leading to refractoriness or relapse in most cases. In these cases, the main objective of treating multiple myeloma is to prolong patient survival and the time until disease progression. Treatment options for relapsed or refractory MM include anti-CD38 or anti-CD319 monoclonal antibodies, steroids, immunomodulatory drugs, proteasome inhibitors, alkylating agents, antimetabolic agents, and transplantation (Cowan et al., 2022; Shah et al., 2012). The treatment of multiple myeloma generates high costs for health systems and operators in the country and worldwide, as the management of the disease is expensive. It is estimated that the annual cost in Brazil is R$124,144 per patient, a value referring to the costs of hospitalization, medications and management of complications (Rodríguez-Otero et al., 2020; Pepe et al., 2018).

Given the current scenario, there is a clear need for new treatment options for patients with multiple myeloma, especially in cases of refractoriness and relapses, in which, often, most of the available treatments have already been used. In this context, immunotherapy, such as the use of CAR-T and/or CAR-NK cells, can provide great help in several aspects (Kõhler et al., 2018; Perez-Amill et al., 2021).

In general, a CAR receptor (chimeric antigen receptor) consists of three linked domains: an extracellular recognition domain, a transmembrane domain, and intracellular signaling domains (also known as the cytoplasmic domain). The extracellular domain is formed by variable portions of the light and heavy chains of an immunoglobulin specific for the recognition of an antigen of choice, optionally linked by a flexible linker (Ramos, C.A., Dotti, G., 2011).

This extracellular portion is linked to a transmembrane domain, which in turn binds to the intracellular signaling domain. The link between the extracellular domain and the transmembrane domain is called the hinge (Miliotou, AN, Papadopoulou, LC, 2018). The intracellular domain can combine signaling domains of the TCR receptor complex and costimulatory molecules of T lymphocytes. These costimulatory molecules are necessary to increase the proliferation, cytotoxicity and persistence of these cells in vitro and in vivo. Thus, CAR cell therapy combines the high specificity of monoclonal antibodies with the potent cytotoxicity and long-term persistence of cytotoxic T cells (Barrett et al., 2014; Zhang et al., 2017). CAR-T cell therapy is a type of treatment in which the patient's T cells are genetically modified so that they become specific and effective in attacking cancer cells. To do this, T lymphocytes are usually collected from the patient and a gene encoding a CAR is inserted into them. This receptor specifically recognizes a protein present in the patient's tumor cells, increasing the affinity of the T lymphocytes (in this case, called CAR-T) for the tumor cells. These cells are then expanded and infused into the patient, where they continue to expand and can recognize and kill cancer cells (Picanto-Castro et al., 2020; Miliotou et al., 2018).

In CAR-NK cell therapy, NK cells from the patient or another donor are genetically modified to attack cancer cells. Similar to T cells, a gene encoding a CAR is inserted into the NK cells, which are then expanded and infused into the patient, where they continue to expand and can recognize and kill cancer cells.

CAR-NK cell therapy emerges as an alternative to CAR-T cells to overcome some limitations such as (i) the difficulty in preparing clinically relevant doses of autologous CAR-T cells in pretreated lymphopenic patients or (ii) the risk of developing graft-versus-host disease (GVHD) associated with the use of T cells collected from an allogeneic source, that is, from another donor (Mehta et al., 2018; Goulmy, E., 1997). NK cells represent the most effective effectors against tumors with a mechanism of action distinct from that of T cells (Davies et al., 2014; Vivier et al., 2012). In contrast to other lymphocytes, NK cells do not express antigen-specific receptors, but rather inactive germline-encoded receptors that are either activators or inhibitors that can induce either a positive or negative signal, and the balance of these signals controls the effector function of NK cells (Lanier, LL, 1998). Mature NK cells also have a relatively limited life span, allowing effective antitumor activity but simultaneously reducing the likelihood of long-term adverse events, such as prolonged cytopenias, due to recognition in non-tumor targets (normal tissues), such as B-cell aplasia (Maude et al., 2014). Furthermore, CAR-NK cells maintain their intrinsic ability to recognize tumor cells through their native receptors. Thus, when compared to T cells in CAR treatment, theoretically, tumor cells are less likely to escape the immune surveillance of NK cells, even if tumor cells downregulate the CAR target antigen (Mehta et al., 2018; Sotillo et al., 2015).

The clinical response of NK cell alloreactivity has been demonstrated in several studies in the setting of hematopoietic stem cell transplantation (HSCT), where patients who received a graft containing alloreactive NK cells had a significantly lower risk of relapse and improved survival (Ruggeri et al., 2002; Cooley et al., 2009; Oevermann et al., 2014; Cooley et al., 2010; Cooley et al., 2014). Adoptive transfer of alloreactive NK cells as stand-alone therapy (independent of HSCT) has also shown encouraging results in a variety of malignancies (Miller et al., 2005; Liopoulou et al., 2010; Rubnitz et al., 2010; Curti et al., 2011; Geller et al., 2011; Bachanova et al., 2014). The success of the antitumor effect of CAR-NK cells has been confirmed in several preclinical studies; however, there are still few clinical studies currently in progress (Liu et al., 2018; Kerbauy et al., 2017).

To date, there are five generations of CAR receptors, which differ from each other in terms of their structure and complexity. The first generation consists of the extracellular domain fused to a CD3 zeta cytoplasmic domain. Although they are effective in vitro, this has not been observed in in vivo assays. Second-generation CARs have an additional costimulatory signaling domain, while third-generation CARs have two or more of these costimulatory domains. The addition of costimulatory domains generates greater proliferation, cytotoxicity, persistence and clinical efficacy (Ramos, CA, Dotti, G., 2011 ; Essand, M., Loskog, ASI, 2013). The addition of two costimulatory domains in ^3rd generation CARs aims to further increase the potency of CAR-T lymphocytes, but trials comparing ^2nd and 3rd ^generation CARs have shown controversial results, so that the superiority of 3rd generation CARs over ^{2nd generation} CARs has not yet been proven. 4th ^generation CARs present the components previously listed with the addition of other genes, such as cytokines, with the aim of increasing the activity of the cells in fighting the tumor. These CARs, once activated, in addition to initiating death mechanisms against the tumor, lead to the production and secretion of cytokines, which act in an autocrine manner, increasing their activation, and also activating other cells of the immune system, thus increasing the antitumor response. Finally, 5th ^generation CARs are currently being developed. They contain the domain of the [3 chain of the cytoplasmic portion of the IL-2 receptor (IL-2R|3), responsible for activating the JAK-STAT pathway in an antigen-dependent manner, leading to the activation and proliferation of T lymphocytes (Wilkins et al., 2017; Till et al., 2012).

Choosing the target antigen for CAR cell therapy is an extremely important step for successful treatment. The ideal target antigen should be strongly expressed in the target cells of the treatment, so that the CAR cells can easily locate the tumor cells in order to eliminate them. In addition, the antigen should be exclusively expressed in the target cells, or have low expression in other cell types. This is important so that the CAR cells do not exert their function in healthy or nonspecific tissues, avoiding adverse effects (Wei et al., 2019).

In the context of multiple myeloma, B cell maturation antigen (BCMA) is the most studied (Picanco-Castro et al., 2020; Barrett et al., 2014; Hay, K. A., Turtle, C. J., 2017; Sermer, D., Brentjens, R., 2019). The BCMA protein belongs to the TNFR superfamily and its expression is restricted to the B cell compartment. Its expression is found in clonal, polyclonal plasma cells and a small portion of inactive germ center B cells, memory cells, and plasmablasts. BCMA expression in cells from patients with multiple myeloma is virtually universal, and in several cases, this protein is overexpressed, making it an ideal target for CAR cell therapy (Rodríguez-Otero et al., 2020; Dogan et al., 2020; Fridman et al., 2018; Shah et al., 2020).

Currently, two anti-BCMA CAR-T cell products are approved by the U.S. Food and Drug Administration (FDA) for use in the treatment of multiple myeloma, namely Idecabtagene-vicleucel and Citacabtagene-autoleucel. The latter was also approved by the Brazilian Health Regulatory Agency (ANVISA) in 2022 for the treatment of patients with relapsed or refractory multiple myeloma who have previously received a proteasome inhibitor, an immunomodulatory agent, and an anti-CD38 antibody (Munshi et al., 2023; Martin et al., 2023).

The anti-BCMA CAR-T products approved for the treatment of multiple myeloma to date are still used as ^the 5th line of treatment, but have potential for use in earlier lines, depending on the improvement of techniques to achieve ideal conditions for treating patients (Cappell, KM, Kochenderfer, JN, 2023).

Several studies and clinical trials have been and continue to be carried out to develop and improve CAR-T and CAR-NK cell therapies. Previous studies have demonstrated excellent therapeutic efficacy, encouraging the continued development of these therapies, and current studies investigate techniques to improve efficacy, reduce toxicity, and mitigate adverse effects (Littman, D., Hexner, E., 2017; Hay, K.A., Turtle, C.J., 2017; Mohanty et al., 2019).

Furthermore, the improvement of techniques for the development and production of CAR-T lymphocytes has been the focus of constant effort, as the methodologies currently available still present difficulties in relation to cell quality, expansion potential, persistence, genetic modifications, among others (Fesnak, AD, 2020; Wang, X., Rivière, I., 2016).

Therefore, there is a clear need for new, improved therapy options for the treatment of multiple myeloma based on CAR-T and/or CAR-NK, which are more efficient and have reduced adverse effects, and which also aim to overcome development and production difficulties.

Based on the above, the objective of the present invention is to provide polynucleotides encoding anti-BCMA CARs with cytotoxic activity for multiple myeloma cells, as well as a vector and an effector immune cell comprising the same, which can be used for the development of more efficient CAR-T and/or CAR-NK cell immunotherapies, seeking to alleviate the operational difficulties and/or adverse effects of the product.

Thus, the inventors have surprisingly managed to overcome the problems of the prior art by means of polynucleotides encoding anti-BCMA chimeric antigen receptors (CARs), in which the CARs comprise an antigen-binding domain, a transmembrane domain and an intracellular domain comprising one or more costimulatory domains and one or more signaling domains, in which the anti-BCMA binding domain comprises the light chain variable domain of SEQ ID NO: 1 and the heavy chain variable domain of SEQ ID NO: 3. The inventors have also developed a vector and a composition, comprising an effector immune cell, comprising the polynucleotide, in addition to a method for the production of a modified effector immune cell and the use of the polynucleotide, the vector, the composition or the effector immune cell produced by the method for the manufacture of a medicament for the treatment of multiple myeloma.

BRIEF DESCRIPTION OF THE INVENTION

The present invention relates to a polynucleotide encoding an anti-BCMA chimeric antigen receptor (CAR) and to a polypeptide corresponding to the anti-BCMA chimeric antigen receptor (CAR) itself. The present invention also relates to a vector and a composition, comprising an effector immune cell, which comprise the polynucleotide.

The present invention is also directed to a method for producing a modified effector immune cell and to the use of the polynucleotide, vector, composition or effector immune cell produced by the method for manufacturing a medicament for the treatment of multiple myeloma.

BRIEF DESCRIPTION OF THE FIGURES

Figures 1 a, 1 b and 1c show the anti-BCMA CAR expression/transfer vectors of the present invention.

Figure 2 shows the packaging cassette plasmid (psPAX2 vector) of the vector of the present invention.

Figure 3 shows the envelope cassette plasmid (pMD2.G vector) of the vector of the present invention.

Figure 4 demonstrates the gating strategy for T lymphocyte immunophenotyping.

Figure 5 shows the results of pre- and post-selection T lymphocyte immunophenotyping with magnetic beads.

Figure 6 is a representative analysis of T lymphocyte activation after stimulation with Dynabeads CD3/CD28 for 24 hours.

Figure 7 demonstrates the gating strategy for CAR+ lymphocyte detection with the BCMA CAR Detection kit.

Figure 8 shows the transduction efficiency of anti-BCMA, anti-BCMA(IL-15), and anti-CD19 CARs.

Figure 9 depicts the viability and expansion of CAR-T lymphocytes in culture for 15 days.

Figure 10 corresponds to the experimental design of the calcein assay. Figure 11 corresponds to the cell lysis curves obtained in the calcein assays of CAR-T cells and non-transduced lymphocytes co-cultured with two tumor cell lines, A) MM1 S (BCMA+ CD19-) and B) Nalm6 (BCMA-CD19+).

Figure 12 demonstrates the gating strategy for cytokine production assay analysis.

Figure 13 shows the production of the cytokines IFN-γ (A) and TNF-α (B) by populations of anti-BCMA CAR-T lymphocytes (with and without IL-15), non-transduced lymphocytes and CD19 CAR-T lymphocytes in response to stimulation by PMA + lonomycin, or when co-cultured with the MM1 S and Nalm6 tumor lines.

Figure 14 represents the gating strategy used for immunophenotyping of NK cells pre- and post-selection.

Figure 15 exemplifies the gating strategy used to analyze NK cells expressing the anti-BCMA-IL-15 CAR.

Figure 16 depicts the transduction efficiency of anti-BCMA-IL-15 CAR for NK and memory-like (ML) NK cells.

Figure 17 shows the transduction efficiency of anti-BCMA-IL-15 CAR for NK and memory-like NK cells.

In Figure 18, the cell death rate for transduced and non-transduced NK and memory-like NK cells co-cultured with the MM1 S cell line can be observed.

Figure 19 shows the gating strategy used to analyze cytokine production by NK and memory-like NK cells expressing the anti-BCMA CAR.

Figure 20 shows the production of IFN-y, TNF-a and CD107a by anti-BCMA-IL-15 NK and NK memory like CAR cells in response to stimulation by PMA + lonomycin, or in co-culture with the MM1 S tumor cell line.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a polynucleotide encoding an anti-BCMA chimeric antigen receptor (CAR), wherein the CAR comprises an anti-BCMA binding domain, a transmembrane domain, and an intracellular domain comprising one or more costimulatory domains and a signaling domain, wherein the anti-BCMA binding domain comprises the light chain variable domain of SEQ ID NO: 1 and the heavy chain variable domain of SEQ ID NO: 3.

The light and heavy chain variable domains of SEQ ID NOs: 1 and 3 correspond to the light and heavy chain variable domains of the rabbit monoclonal anti-murine BCMA antibody, clone 11 D5.3. The light and heavy chain variable domains are encoded by the nucleotide sequences of SEQ ID Nos: 2 and 4, respectively, and degenerate sequences thereof.

In a preferred embodiment, the BCMA-binding variable domains are connected by a flexible peptide linker. In a preferred embodiment, the peptide linker is selected from the group comprising, but not limited to, GSTSGSGKPGSGEGSTKG, (G4S)3, (G4S)4, among others. In a preferred embodiment, the peptide linker is GSTSGSGKPGSGEGSTKG (SEQ ID NO: 5), encoded by SEQ ID NO: 6 and degenerate sequences thereof. In another preferred embodiment, the peptide linker is (G4S)3 (SEQ ID NO: 7), encoded by SEQ ID NO: 8 and degenerate sequences thereof.

In a preferred embodiment, the encoded CAR anti-BCMA binding domain is a Single-Chain Fragment Variable (ScFv), wherein the light and heavy chain variable domains can be in either of the following orientations: light chain variable domain-linker-heavy chain variable domain or heavy chain variable domain-linker-light chain variable domain. In a preferred embodiment, the orientation of the ScFv is light chain variable domain-linker-heavy chain variable domain. In another preferred embodiment, the orientation of the ScFv is heavy chain variable domain-linker-light chain variable domain. In an even more preferred embodiment, the ScFv comprises the sequence of SEQ ID NO: 9, encoded by SEQ ID NO: 10. ID NO: 10 and its degenerate sequences.

In a preferred embodiment, the encoded anti-BCMA CAR includes a transmembrane domain comprising a transmembrane domain of a protein, e.g., selected from the group consisting of the T-cell receptor alpha, beta, or zeta chain, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD 154. In a more preferred embodiment, the encoded transmembrane domain comprises the CD8 transmembrane domain. In an even more preferred embodiment, the encoded transmembrane domain comprises the CD8 alpha transmembrane domain, comprising the sequence of SEQ ID NO: 11, encoded by SEQ ID NO: 12, and degenerate sequences thereof.

In a preferred embodiment, the encoded anti-BCMA binding domain is connected to the transmembrane domain by a hinge region. In a preferred embodiment, the hinge region comprises the constant region of an IgG1 molecule, a CD8 or a CD28 molecule. In a preferred embodiment, the hinge region comprises CD8 alpha. In a more preferred embodiment, the hinge region comprises the sequence of SEQ ID NO: 13, encoded by SEQ ID NO: 14 and degenerate sequences thereof.

In a preferred embodiment, the encoded anti-BCMA CAR includes a signal peptide. In a preferred embodiment, the signal peptide comprises an lgG1 heavy chain signal peptide, a granulocyte-macrophage colony-stimulating factor receptor 2 (GM-CSFR2) signal peptide, or a CD8 alpha signal peptide. In a preferred embodiment, the signal peptide comprises a CD8 alpha signal peptide. In a more preferred embodiment, the signal peptide comprises the sequence of SEQ ID NO: 15, encoded by SEQ ID NO: 16, and degenerate sequences thereof.

In a preferred embodiment, the encoded anti-BCMA CAR includes one or more costimulatory domains to increase cell efficacy and expansion. expressing CAR receptors. As used herein, the term "costimulatory domain" refers to an intracellular signaling domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that provide a second signal necessary for NK/T cell activation and function following antigen binding. In a preferred embodiment, the one or more costimulatory domains are selected from CD28, CD27, OX-40 (CD134), DAP10, DAP 12, 4-1 BB (CD137), CD40L, 2B4, DNAM, CS1, CD48, NKG2D, NKp30, NKp44, NKp46, NKp80, and a combination thereof. In a preferred embodiment, the costimulatory domain comprises 4-1 BB. In a more preferred embodiment, the costimulatory domain comprises the sequence of SEQ ID NO: 17, encoded by SEQ ID NO: 18 and by degenerate sequences thereof. In another preferred embodiment, the costimulatory domain comprises 2B4. In a more preferred embodiment, the costimulatory domain comprises the sequence of SEQ ID NO: 19, encoded by SEQ ID NO: 20 and by degenerate sequences thereof.

In a preferred embodiment, the encoded anti-BCMA CAR includes an intracellular signaling domain that regulates primary activation of the TCR complex in a stimulatory or inhibitory manner. In preferred embodiments, the encoded anti-BCMA CAR comprises a CD3 zeta primary signaling domain. In a more preferred embodiment, the CD3 zeta primary signaling domain comprises the sequence of SEQ ID NO: 21, encoded by SEQ ID NO: 22 and degenerate sequences thereof.

In a preferred embodiment, the encoded anti-BCMA CAR includes a CD3 zeta primary signaling domain and one or more costimulatory signaling domains, which may be linked in any order to the C-terminus of the transmembrane domain.

In a preferred embodiment, the encoded anti-BCMA CAR further includes a cytokine. Cytokines are proteins that regulate the immune response, including, but not limited to, interleukins and interferon-y (IFN-y). Interleukins (IL) are produced by leukocytes in response to microorganisms and other antigens. INF-y is assigned the function of attracting macrophages, which assist in the removal of cellular debris and promote healing and reorganization of areas of inflammation. INF-y is the main cytokine released after induction of the adaptive immune response and is produced by effector T lymphocytes. In a more preferred embodiment, the encoded anti-BCMA CAR includes interleukin, interferon-y (IFN-y) or a combination thereof.

In a more preferred embodiment, the encoded anti-BCMA CAR includes one or more interleukins. Preferably, the encoded anti-BCMA CAR includes IL-2, IL-7, IL-15 or a combination thereof. IL-2 is the main T-cell stimulating factor, as a growth and activation factor for all T lymphocyte subpopulations. IL-7 is a cytokine that stimulates the growth and maturation of B lymphocytes and the activation of T lymphocytes, and is secreted by bone marrow and thymus stromal cells. IL-15 is a cytokine with structural similarity to IL-2 that binds via a complex composed of IL-2/IL-15 receptor beta chain and common gamma chain. IL-15 is secreted by mononuclear phagocytes after virus infection.

In a preferred embodiment, the encoded anti-BCMA CAR includes IL-15. In a more preferred embodiment, the IL-15 cytokine comprises the sequence of SEQ ID NO: 23, encoded by SEQ ID NO: 24 and degenerate sequences thereof. In a more preferred embodiment, the encoded anti-BCMA CAR includes IL-2. In a more preferred embodiment, the IL-2 cytokine comprises the sequence of SEQ ID NO: 25, encoded by SEQ ID NO: 26 and degenerate sequences thereof.

In a preferred embodiment, the encoded anti-BCMA CAR signaling domain is connected to interleukin via a cleavage sequence. In a preferred embodiment, the cleavage sequence comprises 2A sequence elements or 2S-like sequence elements, which can be used for protein binding or co-expression. It is common knowledge in the art that cleavage sequences can be used to co-express genes by linking open reading frames to form a single cistron.

In a preferred embodiment, the cleavage sequence comprised in the encoded anti-BCMA CAR of the present invention comprises equine rhinitis virus (E2A), foot-and-mouth disease virus (F2A), Thosea asigna virus (T2A), porcine Teschovirus-1 (P2A), or a combination thereof. In a preferred embodiment, the cleavage sequence comprises T2A. In a more preferred embodiment, the T2A cleavage sequence comprises the sequence of SEQ ID NO: 27, encoded by SEQ ID NO: 28, and degenerate sequences thereof. In a preferred embodiment, the cleavage sequence comprises P2A. In a more preferred embodiment, the P2A cleavage sequence comprises the sequence of SEQ ID NO: 29, encoded by SEQ ID NO: 30, and degenerate sequences thereof. In a preferred embodiment, the cleavage sequence comprises E2A. In a more preferred embodiment, the E2A cleavage sequence comprises the sequence of SEQ ID NO: 31, encoded by SEQ ID NO: 32 and degenerate sequences thereof.

In a preferred embodiment, the encoded anti-BCMA CAR comprises a CD8 signal peptide, a linker, a CD8 hinge region, a CD8 transmembrane domain, a 4-1 BB costimulatory domain, and a CD3 zeta signaling domain.

In a preferred embodiment, the encoded anti-BCMA CAR comprises a CD8 signal peptide, a linker, a CD8 hinge region, a CD8 transmembrane domain, a 4-1 BB costimulatory domain, a CD3 zeta signaling domain, a T2A cleavage sequence, and IL-15.

In a preferred embodiment, the encoded anti-BCMA CAR comprises a CD8 signal peptide, a linker, a CD8 hinge region, a CD8 transmembrane domain, a 2B4 costimulatory domain, a CD3 zeta signaling domain, a T2A cleavage sequence, and IL-15.

In a preferred embodiment, the encoded anti-BCMA CAR comprises a combination of the sequences selected from the odd sequences of SEQ ID NOs: 1 to 31.

In a preferred embodiment, the encoded anti-BCMA CAR comprises one of SEQ ID NOs: 33, 35 or 37. In a more preferred embodiment, the encoded anti-BCMA CAR comprises SEQ ID NO: 33. In a more preferred embodiment, the encoded anti-BCMA CAR comprises SEQ ID NO: 35. In a more preferred embodiment, the encoded anti-BCMA CAR comprises SEQ ID NO: 37.

In a preferred embodiment, the polynucleotide encoding the anti-BCMA CAR of the present invention comprises a combination of the nucleotide sequences selected from the even sequences of SEQ ID NOs: 2 to 32.

In a preferred embodiment, the polynucleotide encoding the anti-BCMA CAR of the present invention comprises one of SEQ ID NOs: 34, 36 or 38. In a more preferred embodiment, the polynucleotide encoding the anti-BCMA CAR of the present invention comprises SEQ ID NO: 34. In another more preferred embodiment, the polynucleotide encoding the anti-BCMA CAR of the present invention comprises SEQ ID NO: 36. In another more preferred embodiment, the polynucleotide encoding the anti-BCMA CAR of the present invention comprises SEQ ID NO: 38.

The present invention also relates to a polypeptide corresponding to the chimeric antigen receptor (CAR) comprising an anti-BCMA binding domain, a transmembrane domain and an intracellular domain comprising one or more costimulatory domains and a signaling domain, wherein the anti-BCMA binding domain comprises the β-chain variable domain. light chain variable domain of SEQ ID NO: 1 and the light chain variable domain of SEQ ID NO: 3.

The light and heavy chain variable domains of SEQ ID NOs: 1 and 3 of the anti-BCMA CAR of the present invention correspond to the light and heavy chain variable domains of the rabbit monoclonal anti-murine BCMA antibody, clone 11 D5.3.

In a preferred embodiment, the BCMA-binding variable domains of the CAR of the present invention are connected by a flexible peptide linker. In a preferred embodiment, the peptide linker is selected from GSTSGSGKPGSGEGSTKG, (G4S)3, (G4S)4, among others. In a preferred embodiment, the peptide linker is GSTSGSGKPGSGEGSTKG (SEQ ID NO: 5). In another preferred embodiment, the peptide linker is (G4S)3 (SEQ ID NO: 7).

In a preferred embodiment, the anti-BCMA binding domain of the CAR of the present invention is a ScFv, wherein the light and heavy chain variable domains can be in any of the following orientations: light chain variable domain-linker-heavy chain variable domain or heavy chain variable domain-linker-light chain variable domain. In a preferred embodiment, the orientation of the ScFv is light chain variable domain-linker-heavy chain variable domain. In another preferred embodiment, the orientation of the ScFv is heavy chain variable domain-linker-light chain variable domain. In an even more preferred embodiment, the ScFv comprises the sequence of SEQ ID NO: 9.

In a preferred embodiment, the anti-BCMA CAR of the present invention includes a transmembrane domain comprising a transmembrane domain of a protein, e.g., selected from the alpha, beta or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD 154. In a more preferred embodiment, the transmembrane domain of ... of a protein, e.g., selected from the alpha, beta or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD 154. The present invention comprises the CD8 transmembrane domain. In an even more preferred embodiment, the transmembrane domain of the anti-BCMA CAR of the present invention comprises the CD8 alpha transmembrane domain, comprising the sequence of SEQ ID NO: 11.

In a preferred embodiment, the anti-BCMA binding domain of the CAR of the present invention is connected to the transmembrane domain by a hinge region. In a preferred embodiment, the hinge region comprises the constant region of an lgG1 molecule, a CD8 or a CD28 molecule. In a preferred embodiment, the hinge region comprises CD8 alpha. In a more preferred embodiment, the hinge region of the anti-BCMA CAR of the present invention comprises the sequence of SEQ ID NO: 13, encoded by SEQ ID NO: 14.

In a preferred embodiment, the anti-BCMA CAR of the present invention includes a signal peptide. In a preferred embodiment, the signal peptide comprises an lgG1 heavy chain signal peptide, granulocyte-macrophage colony-stimulating factor receptor 2 (GM-CSFR2) signal peptide, or a CD8 alpha signal peptide. In a preferred embodiment, the signal peptide comprises a CD8 alpha signal peptide. In a more preferred embodiment, the signal peptide of the anti-BCMA CAR of the present invention comprises the sequence of SEQ ID NO: 15.

In a preferred embodiment, the anti-BCMA CAR of the present invention includes one or more costimulatory domains to increase the efficacy and expansion of cells expressing the same. In a preferred embodiment, the one or more costimulatory domains of the anti-BCMA CAR of the present invention are selected from, but not limited to, CD28, CD27, OX-40 (CD134), DAP10, DAP 12, 4-1 BB (CD137), CD40L, 2B4, DNAM, CS1, CD48, NKG2D, NKp30, NKp44, NKp46, NKp80, and a combination thereof. In a preferred embodiment, the costimulatory domain of the anti-BCMA CAR of the present invention comprises 4-1 BB. In a more preferred embodiment, the 4-1 BB costimulatory domain comprises sequence of SEQ ID NO: 17. In another preferred embodiment, the costimulatory domain of the anti-BCMA CAR of the present invention comprises 2B4. In a more preferred embodiment, the costimulatory domain 2B4 comprises the sequence of SEQ ID NO: 19.

In a preferred embodiment, the anti-BCMA CAR of the present invention includes an intracellular signaling domain. In preferred embodiments, the anti-BCMA CAR of the present invention comprises a CD3 zeta primary signaling domain. In a more preferred embodiment, the CD3 zeta primary signaling domain comprises the sequence of SEQ ID NO: 21.

In a preferred embodiment, the anti-BCMA CAR of the present invention includes a CD3 zeta primary signaling domain and one or more costimulatory signaling domains, which may be linked in any order to the C-terminus of the transmembrane domain.

In a preferred embodiment, the anti-BCMA CAR of the present invention further includes a cytokine. In a more preferred embodiment, the anti-BCMA CAR of the present invention includes interleukin, interferon-γ (IFN-γ), or a combination thereof.

In a more preferred embodiment, the anti-BCMA CAR of the present invention includes one or more interleukins. Preferably, the CAR of the present invention includes IL-2, IL-7, IL-15 or a combination thereof.

In a preferred embodiment, the anti-BCMA CAR of the present invention includes IL-15. In a more preferred embodiment, the IL-15 cytokine comprises the sequence of SEQ ID NO: 23. In a preferred embodiment, the anti-BCMA CAR includes IL-2. In a more preferred embodiment, the IL-2 cytokine comprises the sequence of SEQ ID NO: 25.

In a preferred embodiment, the signaling domain is connected to the cytokine via a cleavage sequence. In a preferred embodiment, the cleavage sequence comprises sequence elements 2A or 2S-like sequence elements.

In a preferred embodiment, the anti-BCMA CAR of the present invention comprises the cleavage sequence comprising equine rhinitis virus (E2A), foot-and-mouth disease virus (F2A), Thosea asigna virus (T2A), porcine Teschovirus-1 (P2A) or a combination thereof. In a preferred embodiment, the cleavage sequence comprises T2A. In a more preferred embodiment, the T2A cleavage sequence comprises the sequence of SEQ ID NO: 27. In a preferred embodiment, the cleavage sequence comprises P2A. In a more preferred embodiment, the P2A cleavage sequence comprises the sequence of SEQ ID NO: 29. In a preferred embodiment, the cleavage sequence comprises E2A. In a more preferred embodiment, the E2A cleavage sequence comprises the sequence of SEQ ID NO: 31.

In a preferred embodiment, the anti-BCMA CAR of the present invention comprises a CD8 signal peptide, a linker, a CD8 hinge region, a CD8 transmembrane domain, a 4-1 BB costimulatory domain, and a CD3 zeta signaling domain.

In a preferred embodiment, the anti-BCMA CAR of the present invention comprises a CD8 signal peptide, a linker, a CD8 hinge region, a CD8 transmembrane domain, a 4-1 BB costimulatory domain, a CD3 zeta signaling domain, a T2A cleavage sequence, and IL-15.

In a preferred embodiment, the anti-BCMA CAR of the present invention comprises a CD8 signal peptide, a linker, a CD8 hinge region, a CD8 transmembrane domain, a 2B4 costimulatory domain, a CD3 zeta signaling domain, a T2A cleavage sequence, and IL-15.

In a preferred embodiment, the anti-BCMA CAR of the present invention comprises a combination of the sequences selected from the odd sequences of SEQ ID NOs: 1 to 31.

In a preferred embodiment, the anti-BCMA CAR of the present invention comprises one of SEQ ID NOs: 33, 35 or 37. In a more preferred embodiment, the anti-BCMA CAR of the present invention comprises SEQ ID NO: 33. In a more preferred embodiment, the anti-BCMA CAR of the present invention comprises SEQ ID NO: 35. In a more preferred embodiment, the anti-BCMA CAR of the present invention comprises SEQ ID NO: 37.

The present invention also relates to a vector comprising the polynucleotide encoding an anti-BCMA chimeric antigen receptor (CAR), wherein the CAR comprises an anti-BCMA binding domain, a transmembrane domain, and an intracellular domain comprising one or more costimulatory domains and a signaling domain, wherein the anti-BCMA binding domain comprises the light chain variable domain of SEQ ID NO: 1 and the heavy chain variable domain of SEQ ID NO: 3.

In a preferred embodiment, the vector comprises the polynucleotide encoding the anti-BCMA CAR of the present invention, as defined herein.

In a more preferred embodiment, the vector comprises the polynucleotide encoding the anti-BCMA CAR comprising one of SEQ ID NOs: 34, 36 or 38. In a more preferred embodiment, the polynucleotide encoding the anti-BCMA CAR of the present invention comprises SEQ ID NO: 34. In another more preferred embodiment, the polynucleotide encoding the anti-BCMA CAR of the present invention comprises SEQ ID NO: 36. In another more preferred embodiment, the polynucleotide encoding the anti-BCMA CAR of the present invention comprises SEQ ID NO: 38.

In a preferred embodiment, the vector of the present invention is a plasmid or an expression or transfer vector.

In a preferred embodiment, the vector of the present invention comprises one or more promoters. In a preferred embodiment, the one or more promoters are selected from a cytomegalovirus (CMV) promoter, a Rous sarcoma virus (RSV) promoter or a simian virus promoter. (SV40). In a preferred embodiment, the vector of the present invention comprises one or more terminators.

In a preferred embodiment, the vector of the present invention comprises one or more restriction enzyme recognition sites.

In a preferred embodiment, the vector of the present invention comprises one or more regulatory elements. The regulatory elements may comprise, but are not limited to, origins of replication, antibiotic resistance markers, polyadenylation signals, post-transcriptional regulatory elements such as those of the hepatitis B virus (HPRE) or the woodchuck hepatitis virus (WPRE), among others.

In a preferred embodiment, the vector of the present invention is a viral vector. In a preferred embodiment, the vector of the present invention is a viral vector selected from, but not limited to, retroviruses, adenoviruses, and adeno-associated viruses. In a preferred embodiment, the vector of the present invention is an adenovirus. In another preferred embodiment, the vector of the present invention is an adeno-associated virus. In another preferred embodiment, the vector of the present invention is a retrovirus. In a more preferred embodiment, the vector of the present invention is a retrovirus selected from, but not limited to, a lentiviral vector and a gamma-retroviral vector.

In a more preferred embodiment, the vector of the present invention is a gamma-retroviral vector. In another more preferred embodiment, the vector of the present invention is a lentiviral vector. In a preferred embodiment, the lentiviral vector of the present invention is selected from, but not limited to, human immunodeficiency virus (HIV) 1, human immunodeficiency virus (HIV) 2, simian immunodeficiency virus (SIV), bovine immunodeficiency virus (BIV), feline immunodeficiency virus (FIV), etc.

In a preferred embodiment, the vector of the present invention further comprises virus organization genes. In a preferred embodiment, the vector comprises one or more genes encoding structural proteins, enzymes required for replication, viral envelope proteins, accessory proteins, regulatory proteins, and combinations thereof.

In a preferred embodiment, the vector of the present invention is an HIV comprising the virus organization genes, called gag, pol and env. In a preferred embodiment, the vector of the present invention further encodes accessory proteins, such as, but not limited to, Nef, Vpr, Vif and Vpu and regulatory proteins, such as, but not limited to, tat and rev. In a preferred embodiment, the vector of the present invention has specific sequences necessary for signaling processes such as export to the nucleus, signaled by the RRE, integration into the genome and expression, signaled by the Long Terminal Repeats (LTRs), packaging of the RNA into the newly formed virions, signaled by the packaging signal i, and a central polypurine tract (cPPT).

In a preferred embodiment, the vector of the present invention also comprises the Kozak sequence (gccacc).

In a preferred embodiment, the genes for the HIV components are present on a single plasmid. In a preferred embodiment, the genes for the HIV components are present on separate plasmids.

In a preferred embodiment, the vector of the present invention corresponds to a first generation lentiviral vector in which the HIV components are separated into three plasmids. In another preferred embodiment, the vector of the present invention corresponds to a second generation lentiviral vector in which the HIV components are separated into three plasmids and the accessory genes vif, vpr, vpu, and nef have been removed. In another preferred embodiment, the vector of the present invention corresponds to a third generation lentiviral vector in which the HIV components are separated into four plasmids, the vif, vpr, vpu, nef, and tat genes have been removed, and the 5' LTR portion of the transfer plasmid has been replaced by a strong promoter. In a preferred embodiment, the vector of the present invention is a second generation lentiviral vector comprising:

- an expression/transfer vector;

- a packaging cassette plasmid; and

- an envelope cassette plasmid.

In a preferred embodiment, the anti-BCMA CAR expression/transfer vector of the present invention comprises a first, second, third or fourth generation vector. Preferably, the anti-BCMA CAR expression vector of the present invention comprises a second or fourth generation vector. In a more preferred embodiment, the anti-BCMA CAR expression vector of the present invention is selected from, but not limited to, plasmid p4BC, plasmid pB4CI, among others known in the art, which can be designed and synthesized according to practices common to the person skilled in the art. In a preferred embodiment, the anti-BCMA CAR expression vector of the present invention comprises the polynucleotide encoding the light and heavy chain variable domains of clone 11 D5.3.

In a preferred embodiment, the anti-BCMA CAR expression vector of the present invention comprises the polynucleotide as described herein. In a preferred embodiment, the anti-BCMA CAR expression vector of the present invention comprises the nucleotide sequences of SEQ ID NOs: 2 and 4. In a preferred embodiment, the anti-BCMA CAR expression vector of the present invention comprises a combination of two or more nucleotide sequences selected from the even-numbered sequences of SEQ ID NOs: 2 to 32. In a more preferred embodiment, the anti-BCMA CAR expression vector of the present invention comprises any one of SEQ ID NOs: 34, 36 or 38. In a more preferred embodiment, the anti-BCMA CAR expression vector of the present invention further comprises one or more elements common to expression vectors such as one or more promoters, one or more origins of replication, one or more terminators, one or more antibiotic markers, one or more restriction enzyme recognition sites, one or more polyadenylation signals, among others. In a more preferred embodiment, the anti-BCMA CAR expression vector of the present invention further comprises one or more elements common to expression vectors of SEQ ID Nos: 39 to 53.

In a more preferred embodiment, the anti-BCMA CAR expression vector of the present invention comprises any of the vectors as illustrated in Figures 1 a, 1 b and 1 c.

In a preferred embodiment, the packaging cassette plasmid of the present invention is, but is not limited to, the psPAX2 plasmid (Addgene, #12260), which contains the lentiviral gag, pol and RRE genes. The psPAX2 vector is depicted in Figure 2.

In a preferred embodiment, the envelope cassette plasmid is, but is not limited to, plasmid pMD2.G (Addgene, #12259), which contains the envelope coding sequences pseudotyped to the VSV-G virus. The pMD2.G vector is depicted in Figure 3.

The present invention also relates to a composition comprising an effector immune cell comprising the polynucleotide or vector of the present invention as described herein.

In a preferred embodiment, the composition of the present invention is a pharmaceutical composition. The term “pharmaceutical composition” designates a preparation that is in a form that allows the biological activity of an active ingredient contained therein to be effective.

In a preferred embodiment, the composition of the present invention comprises an effector immune cell comprising the polynucleotide encoding an anti-BCMA chimeric antigen receptor (CAR), wherein the CAR comprises an anti-BCMA binding domain, a transmembrane domain and an intracellular domain comprising one or more costimulatory domains and a signaling domain, wherein the anti-BCMA binding domain comprises the light chain variable domain of SEQ ID NO: 1 and the heavy chain variable domain of SEQ ID NO: 3, encoded by the nucleotide sequences of SEQ ID Nos: 2 and 4, respectively, and degenerate sequences thereof.

In another preferred embodiment, the composition of the present invention comprises an effector immune cell comprising the vector. In a more preferred embodiment, the vector comprises an expression vector comprising the polynucleotide encoding the anti-BCMA CAR comprising one of SEQ ID NOs: 34, 36 or 38. In a more preferred embodiment, the polynucleotide encoding the anti-BCMA CAR of the present invention comprises SEQ ID NO: 34. In another more preferred embodiment, the polynucleotide encoding the anti-BCMA CAR of the present invention comprises SEQ ID NO: 36. In another more preferred embodiment, the polynucleotide encoding the anti-BCMA CAR of the present invention comprises SEQ ID NO: 38. In a preferred embodiment, the effector immune cell is transduced with the vector of the present invention.

In a preferred embodiment, the effector immune cell comprises, but is not limited to, T lymphocytes, NK cells, or a combination thereof. In a preferred embodiment, the effector immune cell comprises T lymphocytes. In a preferred embodiment, the effector immune cell comprises NK cells. In a preferred embodiment, the effector immune cell comprises both T lymphocytes and NK cells.

In a preferred embodiment, the effector immune cell is produced by the method of the present invention.

In a preferred embodiment, the composition of the present invention further comprises one or more excipients, carriers or diluents. In a preferred embodiment, the excipients, carriers or diluents are pharmaceutically acceptable excipients known in the art, including, but not limited to, adjuvants, carriers, excipients, glidants, sweetening agents, diluents, preservatives, colorants, flavor enhancers, surfactants, wetting agents, dispersing agents, suspending agents, stabilizers, solvents, emulsifiers acceptable for use in humans or animals. Preferably, the one or more excipients, carriers or diluents comprise sugars, starches, cellulose and its derivatives, gelatin, talc, cocoa butter, waxes, animal and vegetable fats, paraffins, silicones, bentonites, silicic acid, zinc oxide, oils, glycols, polyols, esters, agar; buffering agents, alginic acid, water, saline, Ringer's solution, alcohols, phosphate buffer solutions, and any other compatible substances used in pharmaceutical compositions. Those skilled in the art are highly capable of determining the pharmaceutically acceptable excipients, carriers and diluents for each composition and purpose.

In a preferred embodiment, the composition of the present invention comprises an amount of CAR-expressing immune effector cells contemplated herein. As used herein, the term "amount" refers to "an effective amount" or "a therapeutically effective amount" of a genetically modified cell, e.g., T or NK cell, to achieve a prophylactic or therapeutic result. Those skilled in the art are highly skilled in determining the therapeutically effective amount for each composition and purpose.

In a preferred embodiment, the composition of the present invention may comprise additional agents, such as, but not limited to, cytokines, growth factors, hormones, chemotherapeutics, radiotherapeutics, prodrugs, drugs, antibodies, etc.

Additionally, the present invention relates to a method for producing a modified effector immune cell, comprising introducing into the effector immune cell the polynucleotide or vector of the present invention, as described herein.

In a preferred embodiment, the method of the present invention comprises introducing into the effector immune cell the polynucleotide encoding a anti-BCMA chimeric antigen receptor (CAR), wherein the CAR comprises an anti-BCMA binding domain, a transmembrane domain, and an intracellular domain comprising one or more costimulatory domains and a signaling domain, wherein the anti-BCMA binding domain comprises the light chain variable domain of SEQ ID NO: 1 and the heavy chain variable domain of SEQ ID NO: 3, encoded by the nucleotide sequences of SEQ ID Nos: 2 and 4, respectively, and degenerate sequences thereof.

In another preferred embodiment, the method of the present invention comprises introducing into the effector immune cell the vector. In a more preferred embodiment, the vector comprises an expression vector comprising the polynucleotide encoding the anti-BCMA CAR comprising one of SEQ ID NOs: 34, 36 or 38. In a more preferred embodiment, the polynucleotide encoding the anti-BCMA CAR of the present invention comprises SEQ ID NO: 34. In another more preferred embodiment, the polynucleotide encoding the anti-BCMA CAR of the present invention comprises SEQ ID NO: 36. In another more preferred embodiment, the polynucleotide encoding the anti-BCMA CAR of the present invention comprises SEQ ID NO: 38.

In a preferred embodiment, the effector immune cell comprises, but is not limited to, T lymphocytes, NK cells, or a combination thereof. In a preferred embodiment, the effector immune cell comprises T lymphocytes. In a preferred embodiment, the effector immune cell comprises NK cells. In a preferred embodiment, the effector immune cell comprises both T lymphocytes and NK cells.

In a preferred embodiment, the effector immune cell is transduced with the vector of the present invention. In a preferred embodiment, the vector of the present invention is a lentiviral vector.

In a preferred embodiment, the method of the present invention comprises, prior to the introduction of the polynucleotide or vector of the present invention into the effector immune cell, the transient transfection of the expression vector and the plasmids packaging and envelope cassettes for the production of viral particles.

In a preferred embodiment, transient transfection is done with HEK (Human Embryonic Kidney) cells. In particular, the packaging HEK cells comprise HEK293T.

In a preferred embodiment, the expression vector and the packaging and envelope cassette plasmids are mixed and dripped onto HEK293T cells, which will produce viral particles comprising the polynucleotides of the present invention, wherein the viral particles produced correspond to the vector of the present invention.

In a preferred embodiment, the viral particles will be used for transduction of the effector immune cell of interest.

In a preferred embodiment, the effector immune cell used in the method of the present invention comprises, but is not limited to, T lymphocytes, NK cells, or a combination thereof. In a preferred embodiment, the effector immune cell comprises T lymphocytes. In a preferred embodiment, the effector immune cell comprises NK cells. In a preferred embodiment, the effector immune cell comprises both T lymphocytes and NK cells.

In a preferred embodiment, the source of the immune effector cells used in the method of the present invention is selected from, but not limited to, peripheral blood or umbilical cord blood.

In a preferred embodiment, the method of the present invention comprises, prior to the introduction of the polynucleotide or vector of the present invention into the effector immune cell, the steps of collecting, isolating, selecting and activating the effector immune cells.

In a preferred embodiment, the isolation of the immune effector cells is performed by concentration gradient. Other techniques for isolating the immune effector cells of interest are known in the art and can be used in an alternative way to those described in the present invention.

In a preferred embodiment, selection of immune effector cells is performed through magnetic selection using commercially available kits or beads.

In a preferred embodiment, activation of immune effector cells is performed by means of Dynabeads CD3/CD28 or interleukins appropriate for each cell type, for example, such as interleukins 12, 15 and 18.

In a preferred embodiment, the method of the present invention further comprises a step of identifying the immune effector cells, which can be performed before and/or after the introduction of the polynucleotide or the vector. In a preferred embodiment, the identification of the immune effector cells is performed by immunophenotyping. Other techniques for identifying the immune effector cells are known and can be used in an alternative manner to that described in the present invention.

In a preferred embodiment, the step of introducing the polynucleotide or vector into the immune effector cells of the method of the present invention is carried out by means of transduction.

In a preferred embodiment, transduction comprises the addition of the produced viral particles to the effector immune cells. In a preferred embodiment, following transduction, feeder cells can be added to the culture of the modified effector immune cells.

In a preferred embodiment, the method of the present invention further comprises expanding the modified immune cells.

In a preferred embodiment, the method of the present invention further comprises steps for evaluating the transduction efficiency, viability, expansion, cytotoxicity and cytokine production of the modified immune effector cells.

In a preferred embodiment, the assessment of transduction efficiency is performed using commercial kits that detect CARs directed to the BCMA antigen, followed by flow cytometry. Other techniques are widely known and can be used as alternatives.

In a preferred embodiment, cytotoxicity assessment is performed using the acetoxymethyl calcein (AM) assay, followed by flow cytometry. Other techniques are widely known and can be used alternatively.

In a preferred embodiment, the evaluation of cytokine production is performed by stimulating cells with BCMA positive and negative lines, followed by flow cytometry, also allowing the evaluation of the specificity of the modified immune effector cells.

Additionally, the present invention relates to the use of the polynucleotide, chimeric antigen receptor, vector, composition or effector immune cell produced by the method of the present invention, as described herein, for the manufacture of a medicament for the treatment of multiple myeloma.

DEFINITIONS

Unless otherwise indicated, the terms used in this application should be understood in accordance with conventional usage by those skilled in the art.

The term “peripheral blood mononuclear cell” (PBMC) as used herein refers to cells with a round nucleus, consisting of lymphocytes (T cells, B cells, natural killer (NK) cells) and monocytes, whereas erythrocytes and platelets lack a nucleus, and granulocytes (neutrophils, basophils, and eosinophils) have multilobed nuclei.

The terms “T lymphocyte” and “T cell” as used herein correspond to components of the adaptive immune system whose main function is defense against harmful agents. These cells are derived from hematopoietic stem cells present in the bone marrow and undergo the maturation process in the thymus, where they are lymphocytes that react against antigens presented to them are selected (Famili et al., 2017).

A characteristic that identifies T lymphocytes is the expression of the T cell receptor (TCR), which consists of a transmembrane heterodimer composed of two polypeptide chains covalently linked to each other (mostly a and [3]. The function of the TCR is to recognize presented antigens and, subsequently, activate the T lymphocyte so that it begins its effector functions. The transduction of the activation signal is done by accessory molecules physically linked to the TCR, these being the proteins CD3 and (Mariuzza et al., 2020).

The term “NK cell” as used here refers to natural killer cells or NK cells, a type of lymphocyte necessary for the functioning of the innate immune system due to its cytotoxic activity without the need for prior recognition of a specific antigen, contrary to the functioning of T lymphocytes.

NK cells can be obtained from autologous or allogeneic sources that include peripheral blood, bone marrow, human embryonic stem cells, induced pluripotent stem cells, umbilical cord blood or readily available NK cell lines such as NK92. In a preferred embodiment, the NK cells used in the present invention are obtained from peripheral blood. In another preferred embodiment, the NK cells used in the present invention are derived from umbilical cord (CB). In particular, umbilical cord blood is a readily available allogeneic source that is easily obtained and does not require healthy donors to undergo the risks of collection. The frequencies of NK cells in CB are ~15-20%. To overcome the small blood volume in a CB unit, NK cells have been cultured with irradiated feeder cells, i.e., together with manufactured K562 artificial antigen presenting cells (aAPCs) expressing ligands such as IL-21 and 4-1 BB on the membrane, providing proliferation and activation of NK cells in a reliable and with sufficient doses for adoptive immunotherapy (Shah et al., 2013).

The term “about” used here refers to a value of 5 to 10% more or less than the values to which it refers.

The term “cell viability” as used herein refers to the analysis of metabolically active cells in a cell culture and/or sample in order to assess their activity qualitatively and/or quantitatively. Several techniques for determining cell viability are known in the art and may be used alternatively or concomitantly.

As used herein, the term “lymphocyte expansion” corresponds to the clonal expansion of lymphocytes. Clonal expansion begins with the activation of lymphocytes through the presentation of the antigen of interest by means of the major histocompatibility complex (MHC II) molecules of APCs to T lymphocyte receptors (TCRs). This presentation promoted by APCs is called “stimulation”. Antigen recognition allows the interaction of costimulatory molecule complexes and the secretion of cytokines, which in turn ensure the survival of antigen-specific T cells and activate factors that promote the exposure of specific gene segments, leading to the cellular differentiation of lymphocytes subdivided into subtypes (Th1, Th2 and Th17), which can be identified according to the cytokine they produce the most (INF-γ, IL-4 and IL-21, respectively).

As used here, the term “degenerate sequences” corresponds to nucleotide sequences containing degenerate bases, that is, they encode the same polypeptide due to the redundancy of the genetic code.

As used herein, the term “effector immune cell” corresponds to cells of the immune system that respond to their target cells without the need for co-stimulation, being normally recruited from the circulation, activated by antigens presented by macrophages and secreting cytokines.

The term “drug” as used herein refers to a product suitable for therapy/treatment of multiple myeloma which may comprise, but is not limited to, the polynucleotide encoding the anti-BCMA CAR of the present invention, the vector of the present invention, the CAR of the present invention, the composition of the present invention and the modified effector immune cell produced by the method of the present invention.

In a preferred embodiment, the medicament further comprises one or more excipients, carriers or diluents. In a preferred embodiment, the excipients, carriers or diluents are pharmaceutically acceptable ones known in the art, including, but not limited to, adjuvants, carriers, excipients, glidants, sweetening agents, diluents, preservatives, colorants, flavor enhancers, surfactants, wetting agents, dispersing agents, suspending agents, stabilizers, solvents, emulsifiers acceptable for use in humans or animals. Preferably, the one or more excipients, carriers or diluents comprise sugars, starches, cellulose and its derivatives, gelatin, talc, cocoa butter, waxes, animal and vegetable fats, paraffins, silicones, bentonites, silicic acid, zinc oxide, oils, glycols, polyols, esters, agar; buffering agents, alginic acid, water, saline, Ringer's solution, alcohols, phosphate buffer solutions, and any other compatible substances used in pharmaceutical compositions. Professionals in the field are highly capable of determining the pharmaceutically acceptable excipients, carriers, and diluents for each drug and purpose.

In a preferred embodiment, the medicament comprises a therapeutically effective amount of the polynucleotide, chimeric antigen receptor, vector, method-produced immune effector cell, or composition. Those skilled in the art are highly skilled in determining the therapeutically effective amount of the polynucleotide, chimeric antigen receptor, vector, method-produced immune effector cell, or composition to achieve a prophylactic or therapeutic result for each medicament and intended use. In a preferred embodiment, the medicament may comprise one or more additional agents, such as, but not limited to, cytokines, growth factors, hormones, chemotherapeutics, radiotherapeutics, prodrugs, drugs, antibodies, etc.

In a preferred embodiment, the polynucleotide, the chimeric antigen receptor, the vector, the effector immune cell produced by the method or composition, and the additional agent are contained in a single formulation or in different formulations. In a preferred embodiment, the polynucleotide, the chimeric antigen receptor, the vector, the effector immune cell produced by the method or composition, and the one or more additional agents are contained in a single formulation. In a preferred embodiment, the polynucleotide, the chimeric antigen receptor, the vector, the effector immune cell produced by the method or composition, and the one or more additional agents are contained in different formulations.

In a preferred embodiment, the polynucleotide, the chimeric antigen receptor, the vector, the effector immune cell produced by the method or composition, and the one or more additional agents can be administered together or sequentially.

Additionally, the present invention relates to a method for producing a modified effector immune cell, comprising introducing into the effector immune cell the polynucleotide or vector of the present invention, as described herein.

In a preferred embodiment, the medicament may comprise one or more additional therapeutic agents.

The term “transduction” as used herein refers to the process of transferring DNA or RNA by means of a virus, inspired by the bacterial reproduction process, in which DNA is transferred from one bacterium to another by means of bacteriophages. Preferably, the gene transfer platform for introducing the CAR transgene of the present invention into the immune effector cells is viral-mediated transduction, also called viral expression or transfer vector. Other platforms for gene transfer are known in the art and can be used as alternatives to the technique described here, such as, but not limited to, Sleeping Beauty transposons, piggyBac transposons, or messenger RNA transfection.

As described herein, the viral vector suitable for transducing the CAR transgene of the present invention into immune effector cells is selected from, but not limited to, retroviruses, adenoviruses, and adeno-associated viruses. In a preferred embodiment, the vector of the present invention is an adenovirus. In another preferred embodiment, the vector of the present invention is an adeno-associated virus. In another preferred embodiment, the vector of the present invention is a retrovirus. In a more preferred embodiment, the vector of the present invention is a retrovirus selected from, but not limited to, a lentiviral vector and a gamma-retroviral vector.

In a more preferred embodiment, the vector of the present invention is a gamma-retroviral vector. In another more preferred embodiment, the vector of the present invention is a lentiviral vector. In a preferred embodiment, the lentiviral vector of the present invention is selected from, but not limited to, the human immunodeficiency virus (HIV). Other viral vectors are known in the art and can be used as alternatives to the technique described herein.

The term “transfection” as used herein refers to the process of introducing nucleic acids into eukaryotic cells. Cells can be transfected in a stable manner for the integration of DNA into their genome or in a transient manner for protein expression of temporary duration.

The term “maximum confluence” as used herein corresponds to the maximum percentage of the area of the growth medium covered by adherent cells. For the present invention, the maximum confluence is about 80% to about 100%, preferably about 85%.

The term “immunophenotyping” as used herein refers to the technique laser that evaluates cellular characteristics. Preferably, immunophenotyping is performed by flow cytometry. Other cellular immunophenotyping techniques are known in the art and can be used as an alternative to the technique described here.

Flow cytometry is a technology used to detect and measure physical and chemical aspects of particles. The “gating” strategy corresponds to the basic principle for analyzing the diversity of information provided by the hypotheses tested in flow cytometry, through the refinement and sequential identification of the cell populations under investigation.

EXAMPLES

The following Examples are not intended to limit the scope of the claims of the invention, but rather are intended to be exemplary of certain embodiments. Any variations that occur to one skilled in the art are intended to be included within the scope of the present invention.

EXAMPLE 1 - TRANSIENT TRANSFECTION OF HEK 293T PACKAGING CELLS FOR LENTIVIRAL PRODUCTION

HEK-293T cells (ATCC, CRL-3216) were grown in T75 bottles (Corning, #43064111) with DMEM/F12 medium (Gibco, #11320-033) supplemented with 10% Heat-inactivated Fetal Bovine Serum (HFBS) (Gibco, #16140-071), 1X L-glutamine (Gibco, #25030-081), and 1% penicillin/streptomycin (Gibco, 15070-063) until they reached a maximum confluence of 85% in less than ten passages. If there was an adequate amount of cells, they were detached with trypsin (Gibco, #25200-056), counted and 1.2 x 10 ⁷ cells were plated in a Petri dish (Corning, #430167) with 10 ml of DMEM/F12 medium supplemented so that the confluence was 95-99% on the day of the experiment.

The next day, the plasmid DNA cocktail was prepared in 15 mL conical tubes by adding the following components: in a first tube, 6.92 ug of the plasmid psPAX2 (Addgene, #12260), 3.46 ug of the pMD2.G plasmid (Addgene, #12259), 9.62 μg of the transfer plasmid comprising one of the polynucleotides of the present invention comprising SEQ ID Nos: 34, 36 or 38, and 35 μL of P3000 to a final volume of 1.5 mL of Opti-MEM (Gibco, 31985070) (second generation vectors were used). In another tube, 41 μL of Lipofectamine 3000 (Invitrogen, L3000075) and 1,459 μL of Opti-MEM were mixed. The two solutions were combined and incubated at room temperature for 15 minutes. After the incubation time, all the supernatant was discarded, new 5 ml of supplemented DMEM/F12 were added, the prepared transfection mixture was added drop by drop onto the HEK-293T cells and the plate was incubated in an oven for 6 hours.

After this incubation, the medium was discarded and 10 mL of Opti-MEM medium supplemented with 5% SFBi and 1% penicillin/streptomycin were added, and the plate was again incubated in an oven for 48 hours. After this period, the supernatant containing the viral particles was collected, centrifuged at 300 x g for 5 minutes at 4 °C and filtered through a PES filter (Microlab Scientific, #S33PES045S) with 0.45 μm pores to remove particles and cellular debris from the supernatant containing the lentiviruses. The viral supernatant was aliquoted and stored at -80 °C until transduction of T lymphocytes or NK cells.

EXAMPLE 2 - COLLECTION, ISOLATION AND ACTIVATION OF T CELLS COLLECTION

Healthy volunteers of both sexes were recruited from the Experimental Research Center of the Instituto Israelita de Ensino e Pesquisa (HEP), located at the Hospital Israelita Albert Einstein. All participants signed the Free and Informed Consent Form (FICF).

Peripheral blood collection was performed by venipuncture by the specialized team at Hospital Israelita Albert Einstein. Three tubes of blood (8.8 mL/tube) were collected in a tube with S-Monovette ACD-A (Sarstedt®, NC0504898), to obtain peripheral blood mononuclear cells (PBMC). Peripheral Blood Mononuclear Cell). All samples were alphanumerically identified prior to processing at IIEP to preserve volunteer identification.

Peripheral blood mononuclear cells were obtained using a Ficoll-Paque concentration gradient (Cytiva, #17144003), which separates mononuclear cells from red blood cells and plasma. Blood diluted 1:1 with 1X PBS was added to a conical tube containing Ficoll-Paque, the tubes were centrifuged at 400 x g for 40 min with acceleration set to 4 and deceleration set to 0, and the interphase containing PBMCs was collected and washed with phosphate-buffered saline (PBS) (Gibco, #10010-031).

T cell selection from PBMC was performed by magnetic selection using the Pan T Cell Isolation Kit (Miltenyi, 130-096-535) and the LD column (Miltenyi, 130-042-901), following the manufacturer's instructions. Activation of the selected T cells was performed with anti-CD3/CD28 magnetic beads (Dynabeads, Gibco, 11161 D) at a ratio of 1 bead : 1 cell. The selection and activation steps are also discussed below.

The collection of three tubes of peripheral blood (8.8 mL/tube) yielded, on average, 5.6 x 10 ⁷ PBMC (4.57 x 10 ⁷ - 6.57 x 10 ⁷ , N=5) and negative selection to obtain CD3+ cells yielded, on average, 1.8 x 10 ⁷ cells (7.5 x 10 ⁶ - 4.27 x 10 ⁷ , N=5).

IMMUNOPHENOTYPING OF T-Linphocytes

For immunophenotyping, the cells were analyzed on D0, that is, on the same day that the mononuclear cells were obtained and the T cells were selected.

After cell counting, 5 x 10 ⁵ cells were separated for immunophenotyping by flow cytometry using a panel of 5 markers (Table 1). The remaining cells were maintained in cell culture in RPM1 1640 medium (Gibco, #11875-093) supplemented with 5% Human AB serum (Sigma-Aldrich, H4522), 1% Pen/Strepto and 50 IU of cytokine hll_-2 (Gibco, #PHC0023) - always added fresh). Cells sorted for immunophenotyping were washed twice with 500 pL of FACS Buffer (PBS 1X with 1% SFBi), centrifuged at 300 xg for 5 minutes and resuspended in 100 pL of FACS Buffer. Then, the cells were labeled with 1 pL of LIVE/DEAD viability reagent (Invitrogen™, L34966) and incubated at room temperature for 20 minutes in the dark. After the incubation was finished, two washes were performed with 500 pL of FACS Buffer and the cells were labeled with the antibody pool for immunophenotyping. Table 1 shows the antibody panel used for immunophenotyping and Figure 4 demonstrates the gating strategy used for analysis. For antibody labeling, cells were incubated for 15 minutes at room temperature in the dark with immunophenotyping antibodies. After labeling, cells were washed twice more with FACS Buffer and analyzed using the Attune Nxt cytometer (Thermo Fisher Scientific). Analysis was performed using FlowJo Software v10.6.0.

TABLE 1 - PANEL OF ANTIBODIES USED FOR IMMUNOPHENOTYPING OF

T LYMPHOCYTES.

For the gating strategy, lymphocytes were initially selected from the total population and, among these, singlets (isolated, unitary). Then, cells negative for the viability marker LIVE/DEAD (Invitrogen™) and positive for CD45 ⁺ were selected to compose live leukocytes. Among these, CD3 ⁺ and CD56” cells were selected to exclude NK cells and NKT lymphocytes. Finally, the proportion of CD4 and CD8 T lymphocytes was verified.

Immunophenotyping of pre- and post-selection cells revealed an average of 96.6% (91.5 - 99.8%, N=7) of CD3 ⁺ cells after selection, 62.3% of which were CD4 ⁺ cells (48.5 - 75.8%, N=7) and 32.5% CD8 ⁺ (21.2 - 47.9%, N=7).

SELECTION

Selection with magnetic beads resulted in a population of cells with more than 95% positivity for the T cell marker (CD3+), considered pure for the following experiments. The results of immunophenotyping of T lymphocytes pre- and post-selection with magnetic beads are shown in Figure 5.

ACTIVATION

The selected cells were activated with CD3/CD28 Dynabeads for in vitro maintenance and expansion of the T lymphocyte population. Twenty-four hours after addition of the activation Dynabeads, the activation markers CD25 (BD Pharmigen, 555432) and CD69 (BD Biosciences, #340560) were identified by cytometry (Figure 6).

Figure 6 shows that, before activation with Dynabeads, 94.2% of the cells did not express any of the activation markers. Already 24 hours after activation, approximately 90% of the cells showed expression of at least one activation marker, and almost 85% showed expression of both markers. Thus, it can be concluded that the lymphocytes were properly activated.

EXAMPLE 3 - T CELL TRANSDUCTION

For the production of CAR-T cells, cells were transduced with previously produced lentiviral vectors (e.g., see Example 1). For this, 2.5 x 10 ⁵ activated T cells were seeded per well in 24-well plates in supplemented RPMI culture medium. Then, 1 ml of the lentivirus suspension (non-concentrated) was added. The addition of polybrene at a concentration of 8 pg/mL is considered optional. The plates were homogenized and incubated in a cell culture incubator at 37 °C, with 5% CO 2 and controlled humidity for 16 hours. The following day, the cells were washed and seeded again on the plates in supplemented RPMI. The cells were expanded, maintaining the concentration of 2 x 10 5 cells/mL up to 1 x 106 cells/mL of culture medium, with medium change every 2 days (more frequent when necessary) and transfer to larger bottles as needed.

To evaluate transduction efficiency, transduced cells were labeled with the BCMA CAR Detection Kit (Creative BioLabs, CARD-LX001), which specifically labels CARs directed to the BCMA antigen. The kit was used to detect all CARs of the present invention. To evaluate transduction efficiency, the CD19 CAR Detection kit (Miltenyi, 130-129-550), specific to CARs directed to the CD19 antigen, was also used as a control.

For labeling, 5 × 10 ⁵ cells were collected, had the activation beads magnetically removed, and were then washed, incubated with 1 pL of LIVE/DEAD viability reagent (Invitrogen™) for 20 minutes at room temperature in the dark. Then, the cells were washed with a blocking solution (PBS 1X + 0.5% BSA) in order to reduce nonspecific binding. After washing, 2 pL of BCMA CAR Detection (Creative BioLabs) or CD19 CAR Detection (Miltenyi) detection reagent were added and the cells were incubated for 45 minutes at 4°C in the dark (BCMA CAR Detection) or 10 minutes at room temperature in the dark (CD19 CAR Detection). After incubation, the cells were washed again and labeled with anti-CD3 and anti-CD56 antibodies for 15 minutes at room temperature in the dark. In the case of the CD19 CAR Detection kit, an anti-biotin secondary antibody was also added. Labeled non-transduced T cells were used as a negative control.

Finally, the cells were washed, resuspended in 500 pL of FACS Buffer and acquired on the Attune Nxt cytometer (Thermo Fisher Scientific). The analysis was performed using FlowJo Software v10.6.0. Figure 7 demonstrates the gating strategy used for the analysis of T cells expressing the anti-BCMA CAR.

High transduction efficiency rates were obtained for all

CARs tested (Figure 8). The second-generation CARs, anti-BCMA (of SEQ ID NO: 33, encoded by SEQ ID NO: 34, respectively) and anti-CD19, presented efficiencies higher than 80% (n=2). The anti-BCMA CAR of the present invention comprising IL-15 (SEQ ID NO: 35, encoded by SEQ ID NO: 36, respectively) presented reasonably lower, but still expressive, efficiencies, with an average transduction efficiency of 50% (n=2).

EXAMPLE 4 - T CELL VIABILITY AND EXPANSION

Transduced and non-transduced cells were expanded in vitro for 15 days and monitored for expansion rate and viability (Figure 9).

There were no significant differences in either viability or expansion rate between non-transduced cells and cells transduced with the anti-BCMA CAR vectors of the present invention (n=2). All cells were able to proliferate in culture, with expansion rates of 250 to 400 times in 15 days, and with good viability at the end of in vitro expansion (>90%).

EXAMPLE 5 - T-CELL CYTOTOXICITY ASSAY - CALCEIN AM

Acetoxymethyl calcein (AM) is a dye used to determine the viability of eukaryotic cells. Because it is permeant, calcein passes into living cells, where it fluoresces after hydrolysis of the acetoxymethyl moiety by active intracellular esterases. Dead cells do not have active esterases and therefore do not fluoresce.

The calcein AM cytotoxicity assay was performed using 4 × 10 ⁵ target cells, MM1.S (ATCC, CRL-2974) or Nalm6 (ATCC, CRL-3273) lines, which were labeled with 1 ml of calcein AM (ThermoFisher, C1430) at 25 nM for 30 min in the dark. Then, the cells were washed twice with 10 mL of DPBS + 5% SFBi, resuspended in RPMI medium and 1 × 10 ⁴ cells were plated per well in a 96-well plate (Corning, #3799). Figure 10 corresponds to the experimental design of the calcein assay.

CAR-T or non-transduced lymphocytes were counted and added to the wells containing the target cells in the following ratios: 1:1, 1:2, 2:1, 5:1 and 10:1 (effector cells: target cells). Target cells without calcein labeling, calcein-labeled target cells, and target cells killed with 0.1% triton solution (Sigma-Aldrich) were used as positive and negative controls of the assay. All conditions were acquired by flow cytometry in triplicate.

Effector cells and target cells were incubated for 2 to 4 h in an incubator and, after incubation, readings were taken on the Attune flow cytometer (ThermoFisher), in the channel corresponding to the FITC fluorochrome to identify live target cells (labeled with calcein-AM).

Anti-BCMA CAR-T cells (with and without IL-15) comprising the anti-BCMA CARs of SEQ ID NO: 33 and 35 were co-cultured with MM1 S target cells for two hours and the rate of target cell killing was analyzed. CAR-T cells were also co-cultured with the Nalm6 cell line to verify the specificity of the lymphocyte activity. In addition, co-cultures were performed between non-transduced lymphocytes to verify the basal cytotoxicity of T lymphocytes against the tested cell lines, and between anti-CD19 CAR-T cells to verify the cytotoxicity of genetically modified T lymphocytes in a non-BCMA-specific and CD19-specific manner (Figure 11).

From these results, it is possible to observe that there was a higher death rate of the MM1.S target cells when they were co-cultured with anti-BCMA CAR-T lymphocytes of the present invention comprising the anti-BCMA CARs of SEQ ID NO: 33 and 35, when compared to the co-culture with anti-CD19 CAR-T lymphocytes and non-transduced T lymphocytes (Figure 11 A). Interestingly, the anti-BCMA CAR-T lymphocytes with IL-15 (SEQ ID NO: 35) presented higher cytotoxicity rates compared to the anti-BCMA CAR-T lymphocytes without IL-15 (SEQ ID NO: 33), even though the latter presented a higher percentage of CAR expression.

Furthermore, the anti-BCMA CAR-T lymphocytes of the present invention were more capable of killing MM1.S lineage cells than Nalm6 cells (Figure 11 B), showing the specificity of the cytotoxic action to the BCMA antigen. The analysis of the different proportions of effector cells to target cells allows us to observe that there was a significant increase in the death rates obtained between the smaller proportions (0.5:1, 1:1 and 2:1), however this increase was milder when analyzing the larger proportions (5:1 and 10:1), evidencing a potential plateau in the death potential of the cells.

EXAMPLE 6 - CAR-T CYTOKINE PRODUCTION ASSAY

The cytokine production assay is performed with the aim of identifying the production of the cytokines IFN-y and TNF-a in the anti-BCMA CAR-T lymphocytes comprising the anti-BCMA CAR of SEQ ID NO: 33 and 35, when they were stimulated. For this, four experimental conditions were tested:

(1) a negative control in the absence of stimulation;

(2) a positive control, in which lymphocytes were stimulated with 1X Stimulation Cocktail containing PMA and lonomycin (Invitrogen, #00-4970-93);

(3) stimulation with the MM1 S lineage (BCMA ⁺ ); and

(4) stimulation with the Nalm6 cell line (BCMA-).

In a 96-well plate, 1.5 x 10 ⁵ anti-BCMA CAR-T lymphocytes or non-transduced T lymphocytes, 0.2 pL of Brefeldin A (Invitrogen, #00-4506-51), and 0.75 pL of anti-CD107a antibody (BV785, BD Biosciences) were added per well. In the positive control wells, 1X of the Stimulation Cocktail (Invitrogen) was added, and in the cell stimulation wells, 1.5 x 10 ⁵ MM1.S or Nalm6 cells were added. The conditions were performed in duplicate with a final volume of 200 pL per well and the plate was incubated in an oven for 6 h.

After the incubation time, the cells were passed into cytometry tubes, the wells were washed with 300 pL of FACS Buffer and the contents were added to the respective tubes. The cells were then labeled for CAR receptor identification with the CAR BCMA Detection reagent (Creative BioLabs), washed and then labeled with 1 pL of LIVE/DEAD (Invitrogen™) for 20 minutes. The cells were washed again and incubated with 10 pL and 1.25 pL of the anti-CD3 antibodies. and anti-CD56, respectively, for 15 minutes. Then, the cells were fixed and permeabilized using the Fixation/Permeabilization Kit (BD Biosciences, 554714) and intracellular cytokine labeling was performed with 2.5 pL per sample of anti-IFN-y (PE, BD Biosciences) and anti-TNF-a (PE-Cy7, Biolegend) antibodies. Data acquisition was performed on the Attune cytometer and analysis was performed using FlowJo Software v10.6.0. Below is the antibody panel used (Table 2) and the figure demonstrating the gating strategy used for the analysis is Figure 12.

TABLE 2 - PANEL OF ANTIBODIES USED FOR THE PRODUCTION ASSAY OF

CYTOKINES.

In this assay, an increase in the production of both cytokines analyzed, IFN-y and TNF-a, was observed by anti-BCMA CAR-T lymphocytes when co-cultured with the BCMA+ MM1.S cell line. The same was not observed when the lymphocytes were co-cultured with the BCMA-Nalm6 cell line, reinforcing the specificity of the response to the BCMA antigen. Furthermore, while the production of the cytokine TNF-a was similar between the two anti-BCMA CAR-T lymphocytes (with and without IL-15), the production of IFN-y was greater by lymphocytes with exogenous expression of IL-15 compared to those without exogenous production (Figure 13).

EXAMPLE 7 - CELL SEPARATION, ISOLATION AND ACTIVATION OF NATURAL KILLER (NK) CELLS

UMBILICAL CORD NK CELLS

NK cells were obtained from the umbilical cord of healthy individuals, from the Blood Bank of Hospital Israelita Albert Einstein (HIAE).

After thawing the blood bag, the mononuclear cells are isolated by the density gradient technique (Ficoll Paque Plus, GE Healthcare), as described previously. NK cells are purified from an NK cell isolation kit (NK cell isolation Kit, human - Miltenyi Biotec, 130-092-657) following the manufacturer's instructions.

Subsequently, the cells are co-cultured with K562 feeder cells genetically modified to express IL-21 and 41 BB on their membrane (cells donated by MD Anderson Cancer Center), irradiated at 100 Gy, in a ratio of 2 feeder cells to 1 NK cell (2:1 feeder: NK). The cells are cultured in medium containing RPMI 1640 GLUTAMAX (Gibco, #61870-036) and Click’s medium (Fujifilm Irvine Scientific, #9195) supplemented with inactivated Fetal Bovine Serum (SFBi) (Thermo Fisher Scientific), 1X penicillin/streptomycin and 200 IL/mL of IL-2 (Thermo Fisher Scientific).

PERIPHERAL BLOOD NK CELLS COLLECTION

Peripheral blood collection was performed by venipuncture by the specialized team at Hospital Israelita Albert Einstein. Ten tubes of blood (8.8 mL/tube) were collected in a tube with S-Monovette ACD-A (Sarstedt®) to obtain peripheral blood mononuclear cells (PBMC). All samples were identified alphanumerically before being processed to preserve the identity of the volunteers.

Peripheral blood mononuclear cells were obtained by concentration gradient using Ficoll®-Paque reagent (Cytiva).

For this purpose, peripheral blood is diluted in a 1:1 ratio with sterile PBS, gently homogenized by inversion 5 to 8 times and added to Ficoll®-Paque reagent in a 2:1 diluted blood/Ficoll®-Paque ratio. Subsequently, the tubes containing the diluted blood/Ficoll®-Paque were centrifuged at 650 xg for 40 minutes with acceleration of 4 and deceleration at 0 and the interface containing the PBMC was collected. After PBMC collection, it is centrifuged at 355 xg for 5 minutes to concentrate the cells. Subsequently, the cell concentrate is solubilized in 50 mL of sterile PBS and centrifuged at 355 xg for 5 minutes. After this step, the cell concentrate was solubilized in 15 mL of RPMI 1640 medium containing 10% inactivated fetal bovine serum (BFi) and 1% antibiotic solution (penicillin, 10,000 μg/mL; streptomycin, 10,000 pg/mL).

SELECTION

Selection of NK cells from PBMC was performed by magnetic selection using the human NK Cell Isolation kit (Miltenyi Biotec, 130-092-657) and the LS column (Miltenyi Biotec, 130-042-401). At this step, PBMC cells are first centrifuged at 300 x g for 10 min to obtain the cell concentrate.

The volume of selection reagents was calculated from the total cell count. In the first step, the cell concentrate was resuspended in MACS buffer (Miltenyi Biotec, 130-091-376), dilution 1:20, at a ratio of 40 pL for every 1.0 x 107 cells. Then, NK Cell Biotin-Antibody Cocktail was added at a ratio of 10 pL for every 1.0 x 107 cells and incubated for 5 minutes on ice. Subsequently, MACS buffer, dilution 1:20, at a ratio of 30 pL for every 1.0 x 107 and NK Cell MicroBead Cocktail at a ratio of 20 pL for every 1.0 x 107 were added and incubated for 10 minutes on ice.

After the incubation period, the mixture of cells and components of the selection kit were added to the LS column (Miltenyi Biotec) previously washed with 3 mL of MACS buffer, dilution 1:20, positioned on a support with a magnetic field and the liquid that passed through the column, containing the NK cells, was collected. At this stage, the NK cells were divided into two groups with the same amount of cells, namely: “NK memory like” (ML), NK cells activated with interleukins 12, 15 and 18 and “NK”, NK cells without activation with interleukins. The memory like NK cells were used in this experiment because they presented an improved response when stimulated by cytokines (Romee et al., 2012) and by promoting an increase in the antitumor response (Dong et al., 2022).

ACTIVATION

For activation of ML cells, they were seeded following the proportion of 1.0 X 106 cells/mL in a 24-well plate (Corning, #3473), adding the interleukins IL-12 (10 ng/mL), IL-15 (50 ng/mL) and IL-18 (50 ng/mL) and incubating for 16 hours.

After the incubation time, the ML cells were washed with PBS 1X and centrifuged at 300 x g for 5 minutes. The second wash was performed using RPMI+Click's culture medium + 10% SFB and centrifugation at 300 x g for 5 minutes.

After selection of NK cells or activation of ML cells, these were co-cultured with K562 feeder cells genetically modified to express IL-21 and 41 BB on their membrane (cells donated by MD Anderson Cancer Center), irradiated at 100 Gy, in the ratio of 2 feeder cells to 1 NK cell (2:1 feederNK). The cells were cultured in medium containing RPMI 1640 GLUTAMAX (Thermo Fisher Scientific) and Click’s medium (Fujifilm) supplemented with heat-inactivated Fetal Bovine Serum (SFBi) (Thermo Fisher Scientific), penicillin and streptomycin (P/S) and 200 U/mL of IL-2 (Thermo Fisher Scientific). The PBMC separation and NK selection step were considered day zero (D0) of the experiments.

The collection of ten tubes of peripheral blood (8.8 mL/tube) yielded, on average, 5.90 x 10 ⁷ of PBMC (6.26 x 10 ⁷ - 5.55 x 10 ⁷ , N=2) and the selection to obtain NK cells yielded, on average, 6.7 x 10 ⁶ cells (7.11 x 10 ⁶ - 6.29 x 10 ⁶ , N=2).

IMMUNOPHENOTYPING OF NK CELLS

Immunophenotyping was performed using 5.0 x 105 cells per tube using the flow cytometry technique, using a panel with 3 markers (Table 3).

TABLE 3 - PANEL OF ANTIBODIES USED FOR CELL IMMUNOPHENOTYPING NK

For sample preparation, cells were washed twice with 500 pL of FACS Buffer (PBS 1X with 1% SFBi), centrifuged at 300 x g for 5 minutes, and resuspended in 100 pL of FACS buffer. Cells were then labeled with 1 pL of LIVE/DEAD viability reagent (Invitrogen™) and incubated at room temperature for 20 minutes in the dark. After the incubation time, two washes were performed with 500 pL of FACS Buffer, with centrifugation at 300 x g for 5 minutes, and cells were labeled with the antibody pool for immunophenotyping (Table 3). Labeling was performed for 15 minutes at room temperature, protected from light. After this time, cells were washed twice more with FACS Buffer and acquired on the Attune Nxt cytometer (Thermo Fisher Scientific). Data analysis was performed using FlowJo Software v10.6.0. The gating strategy used for pre- and post-selection NK cell immunophenotyping is shown in Figure 14.

Immunophenotyping of pre- and post-selection cells showed a proportion of 39% of CD3-negative cells in pre-selection and 62% after NK cell selection. CD56-positive and CD16-negative cells represented 29.5% in pre-selection and 31% in post-selection. CD56-positive and CD16-positive cells represented 43.9% in pre-selection and 34.9% in post-selection.

EXAMPLE 8 - NK CELL TRANSDUCTION

NK and ML cells were transduced 6 days after PBMC collection and NK selection (D5) with lentiviral vectors comprising the polynucleotide of SEQ ID NO: 38, produced as described, for example, in Example 1.

Initially during the transduction process, the NK+feeder and ML+feeder cell co-cultures were separated according to the protocol described previously using the human NK Cell Isolation kit (Miltenyi Biotec) and LS column (Miltenyi Biotec).

The transduction process consisted of adding 1 mL of the unconcentrated virus suspension to 24-well plates previously treated with 1 pg/mL of RetroNectin (Takara, T100B) and incubating for 4 hours in an oven at 37 °C, 5% CO2 atmosphere. After the incubation time, 2.5 x 10 ⁵ of NK or ML cells were seeded per well using supplemented RPMI 1640+Click's medium, centrifugation was performed at 1,000 xg for 1 hour at 32 °C and 10% SFBi was added. The following day (D6), the culture medium was changed and feeder cells were added in a 1:1 ratio (feeder: NK).

The evaluation of transduction efficiency occurred 10 days after PBMC collection (D9), using the BCMA CAR detection Kit (Creative BioLabs).

For the labeling process, 2.5 x 10 ⁵ or 5 x 10 ⁵ cells were collected and washed twice with 500 pL of FACS Buffer (PBS 1X with 1% SFBi), centrifuged at 300 xg for 5 minutes and resuspended in 100 pL of FACS buffer. Then, the cells were labeled with 1 pL of LIVE/DEAD viability reagent (Invitrogen™) for 20 minutes at room temperature and in the dark. Then, the cells were washed with a blocking solution (PBS 1X + 0.5% BSA) in order to reduce nonspecific binding. After washing, 2 pL of BCMA CAR Detection reagent (Creative BioLabs) was added and the cells were incubated for 45 minutes at 4°C and in the dark (BCMA CAR Detection). After the incubation time, the cells were washed again and labeled with anti-CD3, anti-CD56, anti-CD16 and anti-CD32 antibodies for 15 minutes at room temperature in the dark. Finally, the cells were washed, resuspended in 500 pL of FACS Buffer and acquired on the Attune Nxt cytometer (Thermo Fisher Scientific). The analysis was performed using FlowJo Software v10.6.0.

Figure 15 exemplifies the gating strategy used for analysis of NK cells expressing the anti-BCMA-IL-15 CAR comprising SEQ ID NO: 37.

In Figure 16, the transduction efficiency for NK and ML cells can be observed. For NK cells, a transduction rate of approximately 7% was observed. For memory-like NK cells, a transduction rate of 10% was observed. In both non-transduced (NT) groups, CAR expression was not detected.

EXAMPLE 9 - VIABILITY AND EXPANSION OF NK CELLS

Transduced and non-transduced cells were maintained in culture for 21 days and monitored for expansion rate and viability.

It was observed that all experimental groups were able to proliferate in culture, with expansion rates between 63 and 104 times in 21 days, maintaining an average viability of 80%. No significant differences were observed between non-transduced cells and cells transduced with the anti-BCMA CAR vectors of the present invention comprising SEQ ID NO: 38 in relation to viability and expansion rate.

The transduction efficiency of anti-BCMA-IL-15 CAR comprising SEQ ID NO: 37 for NK and memory-like NK cells is shown in Figure 17.

EXAMPLE 10 - NK CELL CYTOTOXICITY ASSAY - CALCEIN AM

As discussed above, acetoxymethyl calcein (AM) passes into living cells, where it fluoresces following hydrolysis of the acetoxymethyl moiety mediated by active intracellular esterases. Dead cells do not have active esterases and therefore do not fluoresce.

To verify the cytotoxic effects of transduced and non-transduced NK cells against a BCMA target cell (target of the CAR produced), 4.0 x 10 ⁵ of the MM1.S target cell (ATCC) were labeled with 1 mL of 25 nM calcein AM for 30 minutes in the dark. Then, the target cells were washed twice using 10 mL of DPBS + 5% SFBi, resuspended in supplemented RPMI 1640 medium and plated in a 96-well plate with the amount of 1.0 x 10 ⁴ cells/well.

CAR-NK, CAR-NKML or the respective non-transduced groups were counted and added to the wells containing the target cells at the ratios of 0.5:1, 1:1, 2:1, 5:1 and 10:1 (effector cells:target cells). Target cells without calcein labeling, calcein-labeled target cells and target cells killed with 0.1% triton solution were used as positive and negative controls of the assay. All conditions were acquired by flow cytometry in triplicate.

Effector cells and target cells were incubated for 4 h in an incubator at 37 °C in a CO2 atmosphere and, after the incubation time, acquisition was performed on the Attune flow cytometer, in the channel corresponding to the FITC fluorochrome to identify live target cells (labeled with calcein-AM).

In Figure 18, the cell death rate graphs for NK and ML cells transduced with the anti-BCMA CAR of the present invention comprising SEQ ID NO: 37, encoded by SEQ ID NO: 38, and non-transduced can be observed. The transduced NK and ML cells promoted a higher death rate than the respective non-transduced cells (described in the graphs as NT), with P<0.0001 for the comparison in both groups (comparison between transduced NK and non-transduced NK and transduced ML and non-transduced ML). On average, the difference in the death rate between the transduced and non-transduced groups was 30%.

EXAMPLE 11 - NK CELL CYTOKINE PRODUCTION ASSAY

The cytokine production assay was performed with the aim of identifying the production of the cytokines IFN-y and TNF-a and the expression of CD107a in anti-BCMA CAR-NK and CAR-NKML cells, when these were stimulated in the presence of the target cell MM1 S.

The experiment consisted of three experimental conditions:

(1) a negative control, absence of stimulation; (2) a positive control, in which NK cells were stimulated with a stimulation cocktail (eBioscience™ Cell Stimulation Cocktail, Invitrogen); and

(3) stimulation with the MM1.S lineage (BCMA ⁺ ).

In a 96-well plate, 1.5 x10 ⁵ of anti-BCMA CAR-NK or CAR-NKML cells of the present invention, in which the CAR comprises SEQ ID NO: 37, or their respective non-transduced groups, 0.2 pL of Brefeldin A (Thermo Fisher) and 0.75 pL of the anti-CD107a antibody (BV785, BD Biosciences) were added. In the positive control wells, 1X of the stimulation cocktail (Invitrogen) was added and in the stimulation wells with target cells, 1.5 x 10 ⁵ MM1 S cells were added per well. The conditions were performed in triplicate, with a final volume of 200 pL per well and the plate was incubated in an oven at 37°C, with a CO 2 atmosphere for 6 hours.

After the incubation time, the cells were transferred from the 96-well plate to cytometry tubes, and each experimental well was washed with 300 pL of FACS Buffer. After being transferred to the cytometry tube, the cells were labeled for CAR receptor identification with the CAR BCMA Detection reagent (Creative BioLabs) for 45 minutes, washed and labeled with 1 pL of LIVE/DEAD (Invitrogen™) for 20 minutes.

For labeling with anti-CD3 and anti-CD56 surface antibodies, a wash was performed again and the cells were incubated with 10 pL and 2.5 pL of the antibodies, respectively, for 15 minutes in FACS Buffer, at room temperature.

The cells were then fixed and permeabilized using the Fixation/Permeabilization Kit (BD Biosciences, 554714) and intracellular labeling of anti-IFN-y (PE, BD Biosciences) and anti-TNF-a (PE-Cy7, Biolegend) cytokines was performed using 2.5 pL of antibody per sample. Data acquisition was performed on the Attune Nxt flow cytometer (Thermo Fisher Scientific) and analysis was performed using FlowJo Software v10.6.0. The antibody panel is indicated below. used (Table 4) and the gating strategy used for the analysis (Figure 19).

TABLE 4 - PANEL OF ANTIBODIES USED FOR THE PRODUCTION ASSAY OF

NK CELL CYTOKINES

In this experiment, no significant differences were observed in the production of both cytokines, IFN-y and TNF-a, by anti-BCMA-IL15 CAR-NK NK and ML cells when stimulated by PMA + lonomycin or when co-cultured with the BCMA+MM1 S cell line. No differences were also observed in the production of CD107a for the transduced and non-transduced groups stimulated with PMA + lonomycin or co-cultured with the MM1 S cell line (Figure 20).

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Claims

1. POLYNUCLEOTIDE, characterized by encoding a chimeric antigen receptor (CAR), in which the CAR comprises an anti-BCMA (B cell maturation antigen) binding domain, a transmembrane domain and an intracellular domain comprising one or more costimulatory domains and a signaling domain, in which the anti-BCMA binding domain comprises the light chain variable domain of SEQ ID NO: 1 and the heavy chain variable domain of SEQ ID NO: 3. 2. POLYNUCLEOTIDE, according to claim 1, characterized in that the light and heavy chain variable domains are connected by a linker, preferably a linker of SEQ ID NO: 5. 3. POLYNUCLEOTIDE according to any one of claims 1 to 2, characterized in that the encoded CAR comprises a transmembrane domain selected from the group consisting of T cell receptor alpha, beta or zeta chain, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154, preferably wherein the transmembrane domain is CD8, even more preferably the transmembrane domain is CD8 of SEQ ID NO: 11. 4. POLYNUCLEOTIDE according to any one of claims 1 to 3, characterized in that the encoded anti-BCMA binding domain is connected to the transmembrane domain by a hinge region, preferably wherein the hinge region is CD8, even more preferably the hinge region is CD8 of SEQ ID NO: 13. 5. POLYNUCLEOTIDE, according to any one of claims 1 to 4, characterized in that the encoded CAR further comprises a signal peptide, preferably selected from the group consisting of lgG1 heavy chain signal peptide, granulocyte-macrophage colony-stimulating factor receptor 2 (GM-CSFR2) signal peptide or signal peptide CD8 alpha. 6. POLYNUCLEOTIDE, according to claim 5, characterized in that the signal peptide is CD8, preferably CD8 of SEQ ID NO: 15. 7. POLYNUCLEOTIDE according to any one of claims 1 to 6, characterized in that the one or more costimulatory domains of the encoded CAR are selected from the group consisting of CD28, CD27, OX-40 (CD134), DAP10, DAP 12, 4-1 BB (CD137), CD40L, 2B4, DNAM, CS1, CD48, NKG2D, NKp30, NKp44, NKp46, NKp80 and a combination thereof, preferably wherein the costimulatory domain comprises 4-1 BB and/or 2B4. 8. POLYNUCLEOTIDE according to any one of claims 1 to 7, characterized in that the encoded CAR signaling domain is CD3 zeta, preferably wherein the CD3 zeta signaling domain comprises SEQ ID NO: 21. 9. POLYNUCLEOTIDE, according to any one of claims 1 to 8, characterized in that the encoded CAR further comprises a cytokine, preferably wherein the cytokine is an interleukin (IL). 10. POLYNUCLEOTIDE, according to claim 9, characterized in that the interleukin (IL) is selected from the group consisting of IL-2, IL-7, IL-15 and a combination thereof, preferably IL-15. 11. POLYNUCLEOTIDE, according to any one of claims 9 to 10, characterized in that the cytokine is connected to the CAR signaling domain encoded by a cleavage sequence, preferably in which the cleavage sequence is T2A. 12. POLYNUCLEOTIDE, according to any one of claims 1 to 11, characterized in that the encoded CAR comprises: (a) a CD8 signal peptide, a linker, a CD8 hinge region, a CD8 transmembrane domain, a 4-1 BB costimulatory domain, and a CD3 zeta signaling domain; (b) a CD8 signal peptide, a linker, a CD8 hinge region, a CD8 transmembrane domain, a 4-1 BB costimulatory domain, a CD3 zeta signaling domain, a T2A cleavage sequence, and IL-15; or (c) a CD8 signal peptide, a linker, a CD8 hinge region, a CD8 transmembrane domain, a 2B4 costimulatory domain, a CD3 zeta signaling domain, a T2A cleavage sequence, and IL-15. 13. POLYNUCLEOTIDE, according to any one of claims 1 to 11, characterized by comprising a combination of the nucleotide sequences selected from the even sequences of SEQ ID NOs: 2 to 32. 14. POLYNUCLEOTIDE, according to any one of claims 1 to 13, characterized by comprising any one of SEQ ID NOs: 34, 36 or 38. 15. CHIMERIC ANTIGEN RECEPTOR (CAR) comprising an anti-BCMA (B cell maturation antigen) binding domain, a transmembrane domain and an intracellular domain comprising one or more costimulatory domains and a signaling domain, wherein the anti-BCMA binding domain comprises the light chain variable domain of SEQ ID NO: 1 and the light chain variable domain of SEQ ID NO: 3. 16. RECEPTOR, according to claim 15, characterized in that it is encoded by the polynucleotide, as defined in any one of claims 1 to 14. 17. RECEIVER according to any one of claims 15 to 16, characterized by comprising any one of SEQ ID NOs: 33, 35 or 37. 18. VECTOR, characterized by comprising the polynucleotide, as defined in any one of claims 1 to 14. 19. COMPOSITION, characterized by comprising an effector immune cell that comprises the polynucleotide, as defined in any one of claims 1 to 14, or the vector as defined in claim 18. 20. METHOD FOR PRODUCING A MODIFIED EFFECTOR IMMUNE CELL, characterized by comprising the introduction into the effector immune cell of the polynucleotide, as defined in any one of claims 1 to 14, or of the vector, as defined in claim 18. 21. USE OF THE POLYNUCLEOTIDE, as defined in any one of claims 1 to 14, of the chimeric antigen receptor, as defined in any one of claims 15 to 17, of the vector, as defined in claim 18, of the composition, as defined in claim 19, or of the effector immune cell produced by the method, as defined in claim 20, characterized in that it is for the manufacture of a medicament for the treatment of multiple myeloma.